The suprachiasmatic nucleus (SCN) in the ventral hypothalamus contains the master clock of the brain, controlling the 24-hour circadian rhythm of physiological functions. Recent work has identified in SCN neurons many components of a molecular clock, composed of transcription factors and kinases that interact in regulatory feedback pathways. A major unanswered question is how the molecular clock is connected to the electrical activity of the neurons. The goal of the proposed research is to understand the ionic conductances that control spontaneous firing of SCN neurons and the circadian variation in their firing rate. We will do complementary experiments on intact SCN neurons in brain slice and on a preparation of acutely-dissociated neurons in which pacemaking is maintained. The brain slice preparation allows recordings under the most undisturbed conditions, while dissociated neurons enable voltage-clamp recordings with high time- and voltage-resolution and permit fast solution exchanges and readily reversible application of drugs and channel blockers. Electrophysiological and pharmacological identification of channels underlying various components of electrical current will be complemented by immunocytochemical identification of the channels expressed in the neurons. In addition to characterizing the ionic currents that directly drive pacemaking of SCN neurons, we will characterize other currents that are important for regulating its frequency. We will then determine which of these currents change in amplitude, voltage-dependence, or kinetics to produce the diurnal variation in firing frequency. Finally, we will attempt to discover how the molecular clock is linked to electrical pacemaking and determine whether the electrical clock is regulated by alteration in the activity of kinases or other second messenger pathways. Understanding the mechanisms involved in regulating the excitability of SCN neurons will help in understanding the normal function of the nervous system as well as dysregulation of sleep, attention, and hormonal release.